Project Summary ? Mohler Glioblastoma multiforme (GBM) is the deadliest brain tumor in adults, characterized by rapid progression and poor prognosis due to its highly proliferative and invasive nature. GBM is dynamically heterogeneous with a complex set of inputs ultimately determining a set of outputs (phenotypes) related to cell survival, proliferation, and invasive migration. As a whole, the goals of this project are structured to provide insight into mechanisms of glioblastoma cell migration. The approach is centered on assessing the contributions of underexplored kinase regulatory networks to the phenotypic switch of cancer cells from proliferative to migratory states, underlying aggressive disease progression. As the most abundant post-translational modifications in eukaryotic signaling pathways, protein phosphorylation occupies a central role in regulatory networks and the maintenance of cellular homeostasis, yet their roles in the mediation of cell migration are poorly understood. It has recently been shown that GBM cells can use normal physiological processes for cell migration, such as exploiting ion channels like NKCC1, to promote motility. The Ste20-family kinase SPS1-related proline/alanine-rich kinase (SPAK) relays changes in cell volume to cation-chloride cotransporters (NKCCs and KCCs) to maintain cellular homeostasis and control cell migration. Although previous reports have highlighted the importance of SPAK kinase as a potential therapeutic target for the treatment of GBM, the inability to produce high yields of physiologically phosphorylated kinase, until now, impeded this progress. Leveraging recent advances in orthogonal translation systems and mass spectrometry from the Rinehart lab enables the implementation of a robust pipeline for the identification and validation of lead inhibitor compounds which target physiologically phosphorylated, active SPAK kinase. The impact of candidate SPAK inhibitors on the migration and physiology of GBM cells will be assessed using a microfluidics based 1D cell migration assays. In parallel, the direct interactions and impacts of candidate inhibitor compounds with the GBM proteome will defined using quantitative mass spectrometry techniques. Overall, characterization of small molecule inhibitor compounds identified from this pipeline will provide important mechanistic insight into the role of kinase networks in the regulation of cancer cell migration, expanding potential applications to the broader scope of cancer biology.